白假丝酵母菌体外生物膜药敏性不同检测方法的比较

目的比较不同的方法对白假丝酵母菌体外生物膜药敏性检测的差异。方法分别采用菌落计数法(CFU)、AlamarBlue试剂法、XTT减低法、MTT法对白假丝酵母菌体外48h成熟生物膜的药敏性进行检测,并将AlamarBlue试剂法、XTT减低法、MTT法与CFU法进行比较,观察其相关性及差异性。结果 AlamarBlue试剂法、XTT减低法、MTT法与CFU法都有较高的相关性,相关系数分别为r=0.969、r=0.971、r=0.982(P0.01);与CFU法的差异分别为(0.093±0.127)、(0.054±0.113)、(0.013±0.066),其差异无统计学意义(P0.05)。结论 AlamarBlue试剂法、XTT减低法、MTT法均可替代CFU法对白假丝酵母菌生物膜进行药敏检测。MTT法与CFU法的相关性最大,差异性最小;AlamarBlue试剂作为一种新的试剂,操作简单,对细胞及人类无毒害,更加适合高通量检测。     

可溶性噻唑盐WST-8用于重组人白细胞介素-2的生物学活性测定

运用新型测定活细胞个数的化学物质,建立一种快速测定重组人白细胞介素-2生物学活性的方法。活细胞代谢过程中,胞内的可溶性噻唑盐W ST-8在电子载体1-M ethoxy PMS存在时被还原成可溶性甲臢染料,甲臢染料可在A45Onm处产生吸收值,根据其吸收值的大小间接检测活细胞个数从而测定待检测样品中IL-2的浓度水平。相关性实验结果证明,在细胞浓度1250~160000个/孔范围内,CTLL-2活细胞个数与A45O值之间呈线性相关;用此种新方法检测的白细胞介素-2(IL-2)生物学活性结果与MTT法及XTT法结果基本一致,相关系数分别为r=0.9602和r=0.9511。因此,W ST-8法适用于悬浮依赖型细胞CTLL-2为基础的IL-2生物学活性检测,且具有经济、实用、简便、易行的特点,有较好的实际应用价值。     

雷氏大疣蛛毒液对人肝癌HepG2细胞p21基因表达的影响

本文研究了雷氏大疣蛛毒液对人肝癌细胞株HepG2增殖抑制作用及其分子机制。采用XTT法观察到雷氏大疣蛛毒液剂量依赖抑制HepG2细胞增殖;流式细胞仪检测发现,经过雷氏大疣蛛毒液作用的HepG2细胞周期发生明显的选择性改变;RT-PCR方法检测到p21基因表达增强;Western blot检测发现,p21蛋白表达增加。结果提示,雷氏大疣蛛毒液抑制人肝癌细胞HepG2增殖的可能机制之一是使p21基因和蛋白表达增加,G2IM细胞周期被阻滞,从而诱导细胞凋亡。     

检测人成纤维细胞增殖的XTT比色法

利用3种不同来源的成纤维细胞(大鼠肾上皮细胞,人胎肺成纤维细胞,人成纤维细胞)的活细胞线粒体脱氢酶在电子偶联剂硫酸酚嗪甲酯（phenazine methosulfate, PMS）的协同作用下,还原四氮唑复合物(XTT)形成可溶性的棕黄色甲簪（formazan）产物,测定细胞甲簪的生成量来反映细胞的生长与活性状态,并与传统的四甲基偶氮唑盐(MTT)方法作比较.结果表明,XTT方法直接测定水溶性的甲簪产物,敏感度高于MTT法,具有操作简单、快速、灵敏度高、结果准确的优点,为成纤维细胞的研究建立了新的检测方法.     

白假丝酵母菌突变株ORF19.2500的功能研究

目的筛选50株白假丝酵母菌基因缺失菌,寻找出对白假丝酵母菌生物被膜形成相关基因,进一步探究白假丝酵母菌生物膜的致病机制。方法利用培养生物被膜的方法筛选50株基因缺失菌;利用XTT法验证所筛选出的白假丝酵母菌突变株ORF19.2500生物被膜形成缺陷;进一步观察ORF19.2500基因缺失菌生长、菌丝形成。结果用XTT法证明白假丝酵母菌突变株orf19.2500生物膜形成缺陷,且生长曲线和滴琼脂平板的方法均提示其生长速率减慢。在spider培养基上白假丝酵母菌突变株orf19.2500不能诱导菌丝形成,但在YPD+10%小牛血清则菌丝形成正常。结论白假丝酵母菌突变株orf19.2500可利用spider培养基缺陷而影响其酵母、菌丝二态性的转化,及其生物被膜的形成。     

四唑盐减低法结合激光共聚焦显微镜定量分析表皮葡萄球菌生物被膜体外模型

目的建立体外表皮葡萄球菌(Staphylococcus epidermidis)生物膜(biofilm,BF)模型,观察和定量分析表皮葡萄球菌BF的动态形成过程。方法采用可形成BF的表皮葡萄球菌RP62A,平板法建立体外BF模型,四唑盐(tetrazolium salt,XTT)减低法定量检测BF形成过程中细菌活力的变化,激光共聚焦显微镜(confocal laser scan-ning microscopy,CLSM)结合图像结构分析软件(image structure analyer,ISA)对BF形成过程结构参数进行动态分析,扫描电镜(scanning electron microscope,SEM)观察BF形成过程中的形态结构。结果在12、24和48 h时,XTT减低法A450的值分别为2.39±0.48、3.41±0.18和3.92±0.27,P0.05;ISA软件定量分析显示在区域孔径(AP)的值分别为0.84±0.08、0.68±0.01和0.59±0.13,P0.05,平均扩散距离(ADD)的值分别为1.34±0.24、1.49±0.09和1.89±0.39,P0.05,结构熵(TE)的值分别为4.71±0.82、8.69±0.68和8.94±0.28,24 h、48 h与12 h相比,P0.05。结论表皮葡萄球菌BF的形成是个动态的过程,24 h时BF基本形成,48 h BF结构更加复杂。XTT减低法,CLSM结合ISA软件,SEM三种方法联合使用是观察和定量分析体外BF模型较理想的方法。     

STAT2的小分子干扰RNA抑制HeLa细胞增殖

新近研究发现STAT2基因具有致瘤性.前期研究发现:多种肿瘤组织和细胞系高表达STAT2,因此为进一步研究STAT2基因在肿瘤发生发展中的功能,利用RNA基因沉默技术,降低STAT2基因在宫颈癌HeLa细胞系中的内源表达水平,采用XTT实验、软琼脂集落形成实验以及裸鼠体内成瘤实验等研究策略,发现沉默STAT2基因可抑制...     

铜绿假单胞菌脂多糖对白念珠菌生物膜形成的影响

目的研究铜绿假单胞菌脂多糖(lipopolysaccharide,LPS)对白念珠菌生物膜形成不同时期的影响。方法甲基四氮盐(XTT)减低法用于检测铜绿假单胞菌LPS对白念珠菌生物膜形成不同阶段生成量的影响,利用倒置显微镜观察生物膜形态学改变。结果铜绿假单胞菌对白念珠菌生物膜形成的影响具有阶段差异性和浓度差异性。结论铜绿假单胞菌LPS抑制白念珠菌生物膜菌丝的形成。     

沉默FNBP1基因对人HL-7702细胞体外增殖及迁移能力的影响

为了研究沉默FNBP1(formin-binding protem1)基因对细胞体外增殖以及对细胞迁移能力的影响,设计并构建了3个靶向FNBP1 siRNA干扰载体(Si-1,Si-2,Si-3),经酶切和DNA测序鉴定后转染人正常的肝细胞HL-7702。RT-PCR和Western blot检测瞬时转染细胞中FNBP1基因的干扰效率,筛选出特异高效的RNAi靶点。并用G418筛选稳定表达了RNAi的细胞株,用XTT法检测细胞体外增殖率,划痕法检测细胞的迁移能力。结果显示:酶切和测序结果证明干扰质粒构建正确;转染特异性的干扰质粒(Si-1、Si-2、Si-3)细胞的FNBP1基因表达量与对照组相比均有所降低,在蛋白质水平表达也明显受到抑制,且重组干扰质粒Si-2的干扰效应较强。XTT检测结果表明,构建的沉默FNBP1重组质粒Si-2能显著抑制HL-7702细胞的体外增殖能力,但并不直接影响HL-7702细胞的迁移能力。     

玉米果糖—6—磷酸，2—激酶／果糖—2，6—二磷酸酶基因的结构与表达分析

通过RT-PCR,结果RACE技术,得到了玉米（Zea maysL.)果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶的全长cDNA克隆。命名为mF2KP,氨基酸序列同源性比较发现,mF2KP蛋白可以分为两个部分;C端包含高度保守的催化功能区。N端为植物中特有的多肽,将mF2KP基因中一段包含完整催化功能区的片段在大肠杆菌（Escherichia coli)中表达,融合蛋白具有果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶活性,Northern杂交证明在种子活力不同的幼苗中,mF2KP的转录水平存在明显差异。种子活力越高,幼苗中mF2KP的转录水平越低。     

Requirements for T cell activation by OKT3 monoclonal antibody: role of modulation of T3 molecules and interleukin 1

The requirements for activation of human peripheral blood T cells by the mitogenic monoclonal antibody OKT3 were examined. OKT3 binds to a T cell molecule, T3, associated with the T cell antigen receptor and involved in T cell activation. Activation of T cells by OKT3 requires signals provided by accessory cells and is IL 2 dependent. In the presence of accessory cells, OKT3 induces loss of T3 molecules from the cell surface, production of IL 2, expression of IL 2 receptors, and proliferation. Modulation of T3 molecules by OKT3 can be induced in the absence of accessory cells with anti-mouse IgG. These T cells, however, are not induced to express IL 2 receptors or secrete IL 2. The addition of IL 1 induces expression of IL 2 receptors, but does not induce IL 2 secretion or proliferation. Thus, peripheral blood T cells appear to have different requirements for activation compared with antigen-specific T cell clones that can be induced to produce IL 2 when stimulated with OKT3 and IL 1. Expression of IL 2 receptors does not require modulation of T3 molecules, because the binding of OKT3 to T cells in the presence of IL 1 alone is sufficient to induce IL 2 receptor expression. The results suggest that IL 2 secretion depends on cross-linking and modulation of T3 molecules, and additional, as yet undefined, accessory cell signals. The expression of IL 2 receptors and proliferation of T cells can be induced in the absence of these signals when exogenous IL 2 is provided.     

Characterization of murine T cell activation signals produced by B lymphoma cells

The activation requirements of alloreactive and antigen reactive murine T cells were examined by stimulating class II restricted T cell clones with monoclonal B lymphoma cells. One B lymphoma cell line (T27A) was found to stimulate IL 2 release from some alloreactive T cell clones without stimulating any significant T cell proliferation response. The same B lymphoma cells are capable of stimulating IL 2 release and proliferative responses from other T cell clones. Evidence is presented suggesting that B lymphoma cell stimulation of these T cell clones is largely IL 1 independent and that at least some T cell clones may require activation signals other than Ia, antigen, and IL 1. The addition of exogenous, purified IL 1 to the T cell activation assays was found to have a wide range of stimulatory effects on the proliferative responses of different T cell clones. The absence of comparable IL 1-induced stimulation of IL 2 secretion suggests that IL 1 primarily enhances antigen specific T cell proliferation through mechanisms other than acting as a co-stimulant for IL 2 release.     

TCGF(IL 2)-receptor inducing factor(s). II. Possible role of ATL-derived factor (ADF) on constitutive IL 2 receptor expression of HTLV-I(+) T cell lines

Interleukin 2 (IL 2) receptor (IL 2-R) is constitutively expressed on T cell lines established from the patients with adult T cell leukemia (ATL), which is a human T cell leukemia lymphoma virus (HTLV-1)(+) T4(+)-leukemia endemic in Japan, the United States, and other countries. Many of these cell lines continuously produce an acidic lymphokine, ATL-derived factor (ADF), which preferentially induces the synthesis and expression of IL 2-R on a sensitive HTLV-1(-) non-T cell line (YT). The induced IL 2-R was characterized by the binding of 125I-IL 2 and flow cytometry by using fluoresceinated anti-human IL 2-R monoclonal antibodies (anti-Tac). Scatchard analysis with 125I-IL 2 showed ADF induced high-affinity receptor sites on YT cells. To test the possibility that ADF produced by HTLV-1(+) T cells is involved in the abnormal expression of IL 2-R, we studied the effect of ADF on an HTLV-1(+) IL 2-dependent T cell line (ED) in which the beta-chain gene of the T cell antigen receptor (T beta) was rearranged. Unlike IL 2-independent HTLV-1(+) cell lines that constitutively expressed Il 2-R, the IL 2-R expression on ED cells declined in the absence of crude IL 2 or recombinant IL 2. When either ADF or recombinant IL 2 was added to the culture of ED cells, there was a dose-dependent enhancement of IL 2-R expression in 24 hr. ADF and IL 2 showed a synergism in the IL 2-R induction, and both factors were needed to induce the maximal receptor expression in these T cells. The lack of IL 2 production by ADF-treated YT, as well as ED cell line suggested IL 2 may not be involved in the IL 2-R induction by ADF. Northern blot hybridization with human IL 2-R cDNA probe showed the increase of IL 2-R mRNA in YT cells after ADF-treatment. ADF also enhanced IL 2-R expression of a rat T cell line transformed by HTLV-1(TARS-1), as demonstrated with anti-rat IL 2 receptor monoclonal antibodies (ART-18). An ADF-like IL 2-R-inducing factor was also detected in the conditioned medium of two HTLV-1(+) rat T cell lines (TARL-2 and TART-1), which constitutively expressed a higher number of Il 2-R than TARS-1 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proliferation of T lymphocytes in response to interleukin 2 varies with their state of activation

The interleukin 2 (IL 2) receptor on T lymphocytes can be upregulated by a variety of stimuli including antigen, lectin, and IL 2 itself. In this report, the direct binding of radiolabeled IL 2 and a quantitative bioassay of T cell responsiveness to IL 2 were used to determine the biological significance of upregulation of the murine IL 2 receptor. Antigen and lectin, and to a lesser extent IL 2, were found to cause an increase in the expression of the high affinity form of the IL 2 receptor on both a T cell clone and concanavalin A-induced T cell blasts. A 2-day stimulation with antigen resulted in an increase in the sensitivity of the T cell clone to IL 2, whereas activation with IL 2 caused a decrease in the sensitivity of these cells to subsequent stimulation with IL 2. Comparison of the direct binding and the functional data revealed that IL 2-preactivated T cells required a greater number of occupied high affinity IL 2 receptors to achieve a given fractional response than did unactivated T cells. These observations suggest that the sensitivity with which a T cell responds to IL 2 is not determined solely by the number of high affinity IL 2 receptors it bears.     

Characterization of an immunosuppressive factor derived from colon cancer cells

The colon cancer cell line, HT29, produces a soluble substance (HT29 factor) that blocks mitogen-induced T cell proliferation and the production of interleukin 2 (IL 2). Inhibition of T cell proliferation by the HT29 factor is reversible and is not due to a decline in cell viability or an alteration in the kinetics of T cell proliferation. It occurs even when the HT29 factor is added only 24 hr before terminating the T cell cultures, indicating that the factor affects cell division after activation of T cells has already occurred. No inhibitory activity was found in medium conditioned by human colonic epithelial cells or fibroblasts. The factor has an apparent m.w. of 56,000 and an isoelectric point of 7.9. It is sensitive to endopeptidases, heating to 56 degrees C, and extremes of pH. The HT29 factor also suppresses IL 2 production by T cells. However, low IL 2 availability alone cannot account for the suppressive effect of the factor on T cell proliferation, because the addition of exogenous IL 2 does not reverse the inhibition. This block in IL 2 responsiveness is not primarily due to a decrease in IL 2 receptors because Tac expression on activated T cells is minimally decreased during a 24-hr exposure to the HT29 factor. In addition, IL 2-induced proliferation of mitogen-activated T cells is inhibited only slightly by the HT29 factor, indicating that a block in the interaction of IL 2 with its receptor is not its main mechanism of action. Thus the inhibition of T cell proliferation is likely to be due primarily to a mechanism independent of IL 2.     

Stimulation of immunoglobulin secretion in human B lymphocytes as a direct effect of high concentrations of IL 2

In certain human IgM and IgG cell lines, immunoglobulin (Ig) secretion is highly stimulated by a B cell inducing factor (BIF) that is free of interleukin 2 (IL 2). BIF also induces Ig secretion in purified peripheral blood B cell populations that have been mitogenically stimulated by Staphylococcus aureus bacteria. Low concentrations of IL 2 (less than 20 U/ml) are not active in these systems. We now show that IL 2 at concentrations above 100 U/ml can induce Ig secretion in these blood B cells and B cell lines. Both conventional IL 2, purified from the human JURKAT and gibbon MLA-144 cell lines, and recombinant IL 2 are active. Very high concentrations approaching 10(4) U/ml are optimal for Ig secretion. Antibody to the T cell IL 2 receptor, anti-Tac, did not inhibit stimulation of the IgM cell line SKW6.4 by IL 2, and no Tac antigen was detected on the cells. The 9B11 monoclonal anti-IL 2 antibody that neutralizes T cell growth activity also abrogates stimulation of Ig secretion by conventional and recombinant IL 2 in the SKW6.4 cell line. However, the 1H11 monoclonal anti-(conventional thr3-glycosylated IL 2), which does not neutralize T cell growth activity, does inhibit induction of Ig secretion by the corresponding IL 2 in the B cell line. These results suggest that IL 2 stimulates B cells via a low-affinity interaction with a receptor different from the Tac receptor identified on T cells, and that the active site on the IL 2 molecule for B cells differs from that for T cell targets. If IL 2 promotes Ig secretion by binding with a low affinity to the B cell BIF receptor, IL 2 and BIF could be homologous proteins.     

The role of IL 1 in the antigen-specific activation of murine class II-restricted T lymphocyte clones

The role of IL 1 in the antigen-specific activation of class II-restricted T lymphocytes was examined by using a model system consisting of cloned WEHI 5 B lymphoma accessory cells and class II-restricted, soluble antigen- or alloantigen-reactive T cell clones. The addition of exogenous recombinant IL 1 to the T cell cultures resulted in a significant enhancement of the antigen-specific T cell proliferation response, but at best, only small increases in IL 2 release. Goat IgG anti-IL 1 antibodies were added to the T cell cultures to assess their effect on T cell activation. The IL 1 enhancement of the T cell proliferation response was inhibited by the anti-IL 1 antibodies in a dose-dependent manner. In contrast, only modest levels (10 to 25%) of proliferation inhibition were observed in T cell cultures containing either WEHI 5 or splenocyte accessory cells but no exogenous IL 1. When the anti-IL 1 antibodies were added to primary mixed lymphocyte cultures stimulated by WEHI 5 cells in the absence of exogenous IL 1, no significant inhibition of proliferation was observed. A small but statistically significant proliferation inhibition was observed when anti-IL 1 antibodies were added to mixed lymphocyte reaction cultures stimulated by splenocytes. Two-color cytofluorometric analysis of the effects of IL 1 on antigen-activated T cell clones demonstrated that under suboptimal stimulation conditions, IL 1 stimulated a small but significant increase in the number of T cells bearing IL 2 receptors. In the presence of optimal numbers of WEHI 5 accessory cells, IL 1 enhanced T cell proliferation in the absence of a detectable increase in the number of T cells bearing IL 2 receptors, the number of IL 2 receptors per T cell, or the levels of IL 2 released. Finally, exogenous IL 1 can be added as late as 18 to 24 hr after culture initiation without significantly reducing its ability to enhance the T cell proliferation response. These data indicate that IL 1 has pleiotropic effects on murine T lymphocytes and can function to enhance T cell activation at multiple points during the activation sequence.     

Activation of IL 1-dependent and IL 1-independent T cell lines by calcium ionophore and phorbol ester

We have studied the activation of interleukin 1 (IL 1)-dependent and IL 1-independent T cell lines, specifically their capacity to produce and secrete interleukin 2 (IL 2). The IL 1-dependent T cell lymphoma LBRM33-1A5.47, which requires phytohemagglutinin (PHA) and IL 1 to produce IL 2, was compared with the IL 1-independent T cell lymphoma LBRM33-5A4 and T cell hybridomas DO-11.10/S4.4 and 3DO-54.8. The latter hybridomas do not require exogenous IL 1 to produce IL 2 in response to mitogens or ovalbumin (OVA)/I-Ad. Even though IL 1 is not required by these IL 1-independent T cell lines, we tested whether IL 1 could modulate their response but found no significant effect of exogenous IL 1. We then studied the activation of these T cell lines by the calcium ionophore A23187 and phorbol myristate acetate (PMA). In the case of the IL 1-dependent line LBRM33-1A5.47, there was a strong response when both A23187 and PMA were used simultaneously. We subsequently found that A23187 can replace PHA, and PMA can replace IL 1 in the activation of this cell line to IL 2 production. These observations suggest that the signal(s) provided by PHA and IL 1 involve at least in part a calcium flux, and activation of protein kinase C. Parallel experiments with the use of the IL 1-independent T cell lines showed a strong response to both agents when used simultaneously. A modest response observed to A23187 alone was always enhanced by the addition of PMA. No response was observed to PMA alone. IL 1-rich P388D1 supernatant could replace the enhancing effect of PMA in the response of the IL 1-independent T cell lines. We suggest that the activating signals provided by A23187 and PMA are at least part of the sequence of events that lead to production of IL 2 in either IL 1-dependent or IL 1-independent T cell lines. In IL 1-independent T cell lines, however, both of the activating signals studied may be delivered through stimulation of the Antigen-MHC T cell receptor.     

Mechanism of accessory cell requirement in inducing IL 2 responsiveness by human T4 lymphocytes that generate colonies under PHA stimulation

PHA-driven monoclonal colony formation by low concentrations of resting T4 lymphocytes in agar culture requires the presence of interleukin 2 (IL 2) and accessory cells. Using recombinant IL 2 and anti-Tac monoclonal antibody as a probe for the IL 2 receptor, we demonstrate that the requirement of accessory cells (here an irradiated B cell line) in inducing IL 2 responsiveness relies on their enhancing effect in functional IL 2 receptor expression by the T colony progenitors. Furthermore, it is shown that cell to cell interaction between accessory cells and colony progenitors results in IL 2 response, i.e., colony formation, when the IL 2 receptor density reaches a critical threshold. The asynchronism in IL 2 responsiveness expression by the T colony progenitors upon activation and the short-lived T cell-accessory cell interaction, due to accessory cell death, determine the 10% colony efficiency of the culture system. In addition, we demonstrate that the accessory function in IL 2 receptor and IL 2 responsiveness expression by the T colony progenitors can be supported by irradiated T lymphocytes as well as B cells. The absence of lineage restriction of the signal delivered by accessory cells, and the requirement of physical interaction between T colony progenitors and accessory cells, emphasize the necessity of cross-linking the activation-signal receptors in inducing IL 2 responsiveness by resting T4 cells.     

The tumor promoter teleocidin induces IL 2 receptor expression and IL 2-independent proliferation of human peripheral blood T cells

In testing the recently discovered tumor promoter teleocidin (TCD), we found that like phorbol esters, TCD was mitogenic to human peripheral blood lymphocytes (PBL) and preferentially stimulated sheep erythrocyte-rosetted (ER) T cell-enriched populations. Stimulation of PBL with TCD induced synthesis and expression of receptors for interleukin 2 (IL 2), as shown by dot-blot analysis with the use of a synthetic oligonucleotide probe, cell surface staining with anti-Tac antibody followed by fluorescence-activated cell sorter analysis, and a functional proliferation assay in which TCD-stimulated cells were washed free of TCD and were recultured with human recombinant IL 2 (rIL 2). Increased expression of cell surface markers after TCD stimulation of PBL is not general, because TCD did not affect the expression of Leu-2a antigen, and it also reduced the density of Leu-3a and Leu-4 antigens. Stimulation of cultured, IL 2 receptor-positive PBL with rIL 2, but not TCD, was blocked by anti-rIL 2 antibodies. Furthermore, IL 2-specific mRNA was not detected in TCD-stimulated PBL, demonstrating that IL 2 was not required for TCD-induced T cell proliferation. In addition, TCD replaced IL 2 in inducing short-term proliferation of IL 2-dependent murine cytotoxic T cell lines. The findings that TCD induced IL 2-independent proliferation of T cells, and TCD and IL 2 synergized in inducing T cell proliferation, suggest that they initiate T cell proliferation via different mechanisms. The IL 2-independent activation of T cells, and the induction of IL 2 receptor expression by TCD, may be related to its ability to activate protein kinase C in cell membrane.